Pollack, A. L.*, Barth, A. I. M.*, Altschuler, Y., Nelson, W. J. and Mostov, K. E. (1997). Dynamics of b-Catenin interactions with APC protein regulate epithelial tubulogenesis. Journal of Cell Biology, 137, 1651-1662.

Barth, A. I. M.*, Pollack, A. L.*, Altschuler, Y., Mostov, K. E. and Nelson, W. J. (1997). NH2-terminal deletion of b-catenin results in stable colocalization of mutant b-catenin with Adenomatous Polyposis Coli protein and altered MDCK cell adhesion. Journal of Cell Biology 136, 693-706.

Angres, B., Barth, A. and Nelson, W. J. (1996). Mechanism for Transition from initial to stable cell-cell adhesion: Kinetic analysis of E-cadherin-mediated adhesion using a quantitative adhesion assay. Journal of Cell Biology 134, 549-557.

Barth, A. I. M., Näthke, I. S. and Nelson, W. J. (1997). Cadherins, catenins and APC protein: interplay between cytoskeletal complexes and signaling pathways. Curr. Op. Cell Biol., 9, 683-690.

1. Analysis of signal transduction pathway(s) upstream of beta-catenin that modulate beta-catenin function in response to extracellular signals.

Although growth factors such as HGF induce tyrosine phosphorylation of beta-catenin, and this modification has been associated with a decrease in intercellular adhesion, direct evidence for a regulation of adhesion by beta-catenin remains to be shown. It is also important to analyze the effect of this phosphorylation on the regulation of gene expression by beta-catenin. The results of my postdoctoral research indicate that sequences in the NH2- terminal 90 amino acids of beta-catenin are important for its function in HGF-induced tubulogenesis. The NH2-terminal domain contains several potential recognition sites for tyrosine as well as serine/threonine kinases that may be modified in response to the HGF signal. I have generated MDCK clones expressing full length beta-catenin in which some of these kinase recognition sites are mutated either individually or in combination. These new clones will be analyzed for their phenotypes in intercellular adhesion, migration, HGF-induced tubulogenesis and gene expression, and will be included in the experiments described below. The phosphorylation of these mutant beta-catenins will be also tested in cell lines with decreased or increased activities of certain kinases (as for example the HGF-receptor tyrosine kinase) to compare modulation of beta-catenin by different pathways. The results of these experiments will enable us to identify the kinase(s) immediately upstream of beta-catenin, while the phenotypic analysis of clones expressing these mutant beta-catenins will provide information about the function of a particular phosphorylation.


2. Analysis of the regulation of gene expression by beta-catenin during epithelial morphogenesis.

MDCK clones expressing mutant beta-catenins will be analyzed for the presence and activity of beta-catenin/transcription factor complexes by using reporter plasmid constructs in which transcription of a reporter gene is controlled by a promoter containing the DNA recognition motif for the beta-catenin/transcription factor complex (kindly provided by Drs. Marc van Wetering and Hans Clevers). The regulation of gene expression by different beta-catenin mutant forms and the effect of growth factors on this regulation will be tested with the reporter plasmids. Potential endogenous gene targets of beta-catenin signaling in MDCK cells will be analyzed by mRNA differential display. Expression of mutant beta-catenins in my system is controlled and tightly repressible by tetracycline. This control ensures that differences in gene expression are specific to the induction of beta-catenin signaling, and not caused by clonal variation. This will enable us to screen for differences in the mRNA content of the same clone with or without expression of mutant beta-catenin and thus identify genes involved in epithelial morphogenesis. A long term goal of this approach is to study the role of their gene products in epithelial morphogenesis.


3. Analysis of the regulation of cell adhesion by beta-catenin.

The regulation of cell adhesion is a key factor during epithelial morphogenesis. I will investigate how growth factor-induced phosphorylation of beta-catenin regulates adhesion. In collaboration with Dr. Brigitte Angres, I have adapted the adhesion assay developed by Dr. David McClay to specifically measure E-cadherin mediated intercellular adhesion (Angres et al., 1996). This centrifugal force-based adhesion assay is a useful tool to measure the effect of growth factors, such as HGF, or of the expression of mutant beta-catenins on E-cadherin mediated adhesion. This assay will be used to analyze the effect of different signal transduction pathways and of beta-catenin phosphorylation on E-cadherin mediated adhesion. In addition, MDCK clones expressing mutant forms of E-cadherin will be analyzed using this adhesion assay and compared to the clones expressing mutant beta-catenin in their ability to undergo HGF-induced tubulogenesis.


4. Analysis of the role of the APC protein/beta-catenin complex in morphogenetic movements of epithelial cells using GFP fusion proteins.

The results of my research suggest that microtubule-associated APC protein/beta-catenin complexes are involved in regulating cell motility. In order to investigate this hypothesis, I will analyze the dynamics of microtubules which are associated with the APC protein clusters. In addition, I will study the regulation of these dynamics by beta-catenin. In collaboration with Dr. Eugenio de Hostos (Rice University, Houston), I have expressed fusions of green fluorescent protein (GFP) with tubulin, actin, and with both wild-type and mutant beta-catenin, in MDCK cells. Currently, we are also generating an expression vector for GFP-APC and these fusion proteins will be used to study the roles of APC protein and beta-catenin in motility and signaling. In collaboration with Mrs. Cynthia Adams and Dr. Stephen Smith (Stanford University), I have begun to analyze the dynamics of the actin- and microtubule-associated beta-catenin complexes in migrating MDCK cells using a scanning laser confocal and DIC microscope equipped for time-lapse recording. I intend to set up a similar microscopy workstation in my future laboratory to continue these experiments. The same microscopic techniques will be used to study the dynamics of these GFP-fusion proteins during tubulogenesis. See Movie Page.


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